Project Summary Temporomandibular joint disorders (TMJDs) are estimated to affect over 10 million Americans, with ~70% of TMJDs patients suffering from displacement of the TMJ disc as per NIDCR. Total 80 - 90% of symptomatic TMJDs patients have internal derangement (ID), also referred to as disc displacement, which is highly associated with osteoarthritis (OA) that may necessitate surgical treatment. Previous attempts to replace the TMJ disc with alloplastic and/or synthetic grafts have failed, resulting in further joint degradation. Thus, regeneration of TMJ disc has recently emerged as an alternative approach to overcome limitations of current treatments for TMJ disorders. In our preliminary study, we developed anatomically correct 3D-printed bioscaffolds with native-like microfiber orientation and micro-precise spatiotemporal delivery of growth factors. Briefly, scaffold was consisted of polycaprolactone (PCL) microstrands, circumferentially oriented in the peripheral bands and aligned from anterior to posterior in the intermediate zones, mimicking the region- dependent collagen orientation in native TMJ discs. Microstrands and microchannels were tuned to match the mechanical properties with those of native tissues. To guide formation of regionally distributed fibrous and cartilaginous matrix in TMJ discs, connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGF?3) encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (?S) were spatially embedded in scaffold?s microstrands. Upon 4 wks in vitro culture with TMJ synovial mesenchymal stem/progenitor cells (syMSCs), heterogeneous fibrocartilaginous tissue was formed with overall distribution of collagen-rich fibrous matrix and fibrocartilaginous matrix in intermediate zone, reminiscent of the native feature of TMJ disc. In rabbits, CTGF/TGF?3 ?S-embedded scaffolds successfully led to regeneration of perforated TMJ disc and protected articular surface. Accordingly, the overall objectives are to establish an efficient strategy for regeneration of TMJ disc with anatomically correct scaffolds from stem/progenitor cells. Our overarching hypothesis is that spatiotemporal delivery of CTGF and TGF?3 in 3D-printed scaffolds with biomimetic microstructure leads to regeneration of heterogeneous and anisotropic TMJ disc from endogenous mesenchymal stem/progenitor cells. As an inevitable and timely step in our pathways for TMJ disc regeneration, we propose to determine effective doses and release rates of CTGF and TGF?3 for engineering TMJ disc and to investigate effects of 3D-printed bioscaffolds on TMJ disc regeneration in a pre-clinical large animal model. The proposed approach may serve as an effective approach to regenerate TMJ discs.